Objective (A) Arenaviruses: Lassa virus isolates from Mali are less virulent then classical isolates, and in macaques disease progression was atypical with primarily pulmonary disease manifestation. Our results suggest that clinical presentation of Lassa fever in Mali may be atypical, which might explain the low incidence of disease in this region. Bunyaviruses: We have characterized two animal models for the study of Sin nombre virus; an infection model in Syrian hamsters and a disease model in rhesus macaques. Using the novel nonhuman primate model we performed the first temporal characterization of the pathophysiology of hantavirus pulmonary syndrome and identified several key factors, including viremia, hematological abnormalities and tissue specific immune dysregulation in the development of this rare but frequently fatal disease. Filoviruses: The Syrian hamster we have developed displays almost all hallmark features of human disease including coagulopathy, and the suitability for antiviral studies seems promising. Using this model, we have investigated the mechanisms in which hamsters are protected from wild-type Ebola virus and determined that a CD4+ T cell response, facilitating a neutralizing antibody response is responsible for natural immunity (unpublished data). Paramyxoviruses: We have performed a histopathological comparison of two strains of Nipah virus in the Syrian hamster model, representing the two proposed genotypes of Nipah virus, Malaysia and Bangladesh. No difference in severity of rhinitis and bronchointerstitial pneumonia could be detected on 4 days after inoculation, although these lesions seemed slightly more severe in animals infected with the isolate from Bangladesh on 2 days after inoculation. Moreover, we gained some insight in the tropism of Nipah virus by showing that Nipah virus antigen could only be detected in small and medium size arteries but not veins (unpublished data). Objective (B) Bunyaviruses: Ongoing transfer and depletion studies using our adenovirus-based CCHF vaccines expressing the nucleoprotein or glycoproteins of CCHF virus indicated that antibodies are the mechanism of protection of these vaccine vectors. We have also performed a study to examine the durability of the VSV-based Andes virus vaccine in hamsters. This vaccine afforded significant protection when given 6 months prior to challenge, but lost efficacy after 1 year. Filoviruses: We have investigated the mechanism of protection of the recombinant Vesicular stomatitis virus (rVSV) based Ebola vaccine in nonhuman primates. Depletion studies demonstrated that neutralizing antibody responses are critical for the efficacy of the vaccine (Marzi 2013). Furthermore, we have evaluated the protective efficacy of live or inactivated rabies-based Ebola vaccines expressing the Zaire ebolavirus glycoprotein in a nonhuman primate model. Protection was achieved with all vaccination approaches using a prime/boost strategy and largely dependent on the quality of the humoral immune response. All our filovirus vaccine efforts were summarized and discussed in several recent review articles. Paramyxoviruses: We developed rVSV-based Nipah virus vaccines expressing either the nucleoprotein (N), fusion protein (F) or glycoprotein (G) of Nipah virus, strain Malaysia. Efficacy studies were performed in the Syrian hamster model and showed that the F and G protein expressing vaccine vectors provide full protection in this animal model. Current studies support the notion that the humoral immune responses are critical for vaccine efficacy. The vaccine was also tested in the African green monkey model and was efficacious in protecting animals from Nipah virus disease (Prescott in preparation). Furthermore, this vaccine protects hamsters when administered within one day of challenge and be utilized for ring vaccination in outbreak settings. Objective (C) Bunyaviruses: We are currently characterizing the immune response to hantaviruses in their natural hosts. For this we have been using our in-house deer mouse colony, the reservoir for Sin nombre virus (North American hantavirus). In contrast to Sin nombre, which persistently infects deer mice, Andes virus infection is efficiently cleared by deer mice. Deer mice mount an anti-inflammatory response to Sin nombre virus allowing for persistence, but mount a strong inflammatory response to Andes virus. It is thought that hantaviruses cause disease via an immunopathogenic mechanism. For this, we performed T cell depletion in hamsters, a model for human disease. Depletion did not alter the pathogenicity or replication of Andes virus indicating that pro-inflammatory T cell responses do not contribute to disease. Paramyxoviruses: In Bangladesh, outbreaks of Nipah virus occur almost every year, resulting in respiratory and neurological disease with high case-fatality rates. Based on epidemiological data Nipah virus is thought to be transmitted from fruit bats to humans via drinking of date palm sap contaminated by bats. Additionally, human-to-human transmission has been shown to occur. We experimentally modeled the proposed transmission cycle of Nipah virus in Syrian hamsters. Hamsters that drank artificial palm sap containing Nipah virus became infected and developed neurological signs of disease. Virus replication occurred mainly in the respiratory tract. Hamsters infected with Nipah virus through drinking of contaminated palm sap could transmit the virus to uninfected cage mates. Objective (D) Filoviruses: Ribavirin can effectively extend the time-to-euthanasia of hamsters infected with Ebola virus, but as observed in cell culture appears to rapidly develop resistance that is either viral or cellular based. DEB025 has shown activity against Ebola virus in vitro (EC50 5uM) and combination treatment with ribavirin has been assessed; however a synergic antiviral effect was not observed. We have also screened a small collection of broad spectrum antivirals that are in development against influenza and other RNA viruses using a recombinant Ebola that expresses Firefly luciferase generating two potential lead compounds for further assessment. In the past year we have further utilized recombinant Ebola viruses to study virus biology, and identified the modulation of TGF-&#946; signaling by Ebola virus and developed a model for its role in pathogenesis, as well as in analyzing the role of gene borders on transcriptional regulation of Ebola virus gene expression. Furthermore, we have developed novel experimental systems that allow modeling of the complete Ebola virus life cycle over multiple infectious cycles without the need for a high containment laboratory. We will continue this work, and are now in the process of moving this into animal models to study pathogenesis. We have studied the mechanism of Ebola virus RNA editing. Studies have identified primary and secondary structural requirements and a potential role for VP30 as a necessary factor for RNA editing. Objective (E) Arenaviruses: Our continuous ecological studies have further defined endemic areas for a unique Lassa virus, Soromba R, in Mali. In conjunction, we have begun breeding Mastomys natalensis, the natural reservoir of Lassa virus. First infection studies defined the animals as susceptible to Lassa virus.